RNase-stable RNA: conformational parameters of the nucleic acid backbone for binding to RNase T1.

An RNA sequence showing high stability with respect to digestion by ribonuclease T1 (RNase T1) was isolated by in vitro selection from an RNA library. Although ribonuclease T1 cleaves single-stranded RNA specifically after guanosine residues, secondary structure calculations predict several guanosines in single-stranded areas. Two of these guanosines are part of a GGCA-tetraloop, a recurring structure element in the secondary structure predictions. Molecular dynamics simulations of the conformation space of the nucleotides involved in this tetraloop show on the one hand that the nucleic acid backbone of the guanosines cannot realise the conformation required for cleavage by RNase T1. On the other hand, it could be shown that an RNA molecule not forced into a tetraloop occupies this conformation several times in the course of the simulation. The simulations confirm the GGCA-tetraloop as an RNase-stable secondary structure element. Our results show that, besides the known prerequisite of a single-stranded RNA, RNase T1 has additional demands on the substrate conformation.     

Characterization of nucleic acids in membrane vesicles from scrapie-infected hamster brain.

This study reports the partial characterization of nucleic acids present in gradient fractions enriched for large membrane vesicles from scrapie-infected and uninfected hamster brains. Labeling of phenol-extracted nucleic acids at the 3' or 5' ends revealed abundant amounts of low-molecular-weight RNA and little or no DNA. These nucleic acids survived nuclease treatment of membrane vesicles but were sensitive to RNase after phenol extraction. Analysis of 5'-end-labeled nucleic acids by one- and two-dimensional gel electrophoresis revealed an RNA of ca. 100 bases in preparations from scrapie-infected hamster brain that could not be detected in uninfected brain. The possibility that this apparently unique small RNA may result from tissue damage or abnormal RNA processing or may be a component of the infectious complex is discussed.     

Highly chlorinated Escherichia coli cannot be stained by propidium iodide

Several studies have shown that the staining by fluorochromes (DAPI, SYBR Green II, and TOTO-1) of bacteria is altered by chlorination. To evaluate the effect of chlorine (bleach solution) on propidium iodide (PI) staining, we studied Escherichia coli in suspension and biomolecules in solution (DNA, RNA, BSA, palmitic acid, and dextran) first subjected to chlorine and then neutralized by sodium thiosulphate. The suspensions and solutions were subsequently stained with PI. The fluorescence intensity of the PI-stained DNA and RNA in solution dramatically decreased with an increase in the chlorine concentration applied. These results explain the fact that for chlorine concentrations higher than 3 micromol/L Cl2, the E. coli cells were too damaged to be properly stained by PI. In the case of highly chlorinated bacteria, it was impossible to distinguish healthy cells (with a PI-impermeable membrane and undamaged nucleic acids), which were nonfluorescent after PI staining, from cells severely injured by chlorine (with a PI-permeable membrane and damaged nucleic acids) that were also nonfluorescent, as PI penetrated but did not stain chlorinated nucleic acids. Our results suggest that it would be prudent to be cautious in interpreting the results of PI staining, as PI false-negative cells (cells with compromised membranes but not stained by PI because of nucleic acid damage caused by chlorine) are obtained as a result of nucleic acid damage, leading to an underestimation of truly dead bacteria.     

In situ analysis of nucleic acids in cold-induced nonculturable Vibrio vulnificus.

Low-temperature-induced nonculturable cells of the human pathogenic bacterium Vibrio vulnificus retained significant amounts of nucleic acids for more than 5 months. Upon permeabilization of fixed cells, however, an increasing number of cold-incubated cells released the nucleic acids. This indicates substantial degradation of DNA and RNA in nonculturable cells prior to fixation. Treatment of permeabilized cells with DNase and RNase allowed differential staining of DNA and RNA with the nucleic acid dye 4',6-diamidino-2-phenylindole (DAPI). Epifluorescence microscopy revealed that the could-induced nonculturable populations of V. vulnificus are highly heterogeneous with regard to their nucleic acid content. The fraction of nonculturable cells which maintained DNA and RNA structures decreased gradually during cold incubation. After 5 months at 5 degrees C, less than 0.05% of the cells could be observed to retain DNA and RNA. In parallel with the loss of nucleic acids, an increase in the concentrations of UV-absorbing material in the culture supernatants was observed in nonculturable-cell suspensions. It is hypothesized that there are two phases of the formation of nonculturable cells of V. vulnificus: the first involves a loss of culturability with maintenance of cellular integrity and intact RNA and DNA (and thus possibly viability), and the second is typified by a gradual degradation of nucleic acids, the products of which partly remain inside the cells and partly diffuse into the extracellular space. A small number of nonculturable cells, however, retain DNA and RNA, and thus may be viable despite having reduced culturability.     

Nucleic acids potentiate Factor VII-activating protease (FSAP)-mediated cleavage of platelet-derived growth factor-BB and inhibition of vascular smooth muscle cell proliferation

FSAP (Factor VII-activating protease) can cleave and inactivate PDGF-BB (platelet-derived growth factor-BB) and thereby inhibits VSMC (vascular smooth-muscle cell) proliferation. The auto-activation of FSAP is facilitated by negatively charged polyanions such as heparin, dextransulfate or extracellular ribonucleic acids. Since auto-activation is essential for the anti-proliferative function of FSAP, the influence of nucleic acids as cofactors for the FSAP-mediated inhibition of PDGF-BB was investigated. Natural or artificial RNA was an effective cofactor for FSAP mediated PDGF-BB degradation, whereas the effect of DNA was weak. RNA-induced cleavage of PDGF-BB was inhibited by serine protease inhibitors. The pattern of PDGF-BB cleavage was identical with either heparin or RNA as a cofactor. One of the cleavage sites in PDGF-BB was at the positions 160-162 (R160KK162), which is an important region for receptor binding and activation. In VSMCs, PDGF-BB-stimulated DNA synthesis was inhibited by FSAP in the presence of RNA. RNA was more effective than DNA and the cofactor activity of RNA was neutralized after pretreatment with RNase. FSAP binding to RNA protected the nucleic acid from degradation by RNase. These data are relevant to situations where extracellular nucleic acids released from necrotic or apoptotic cells could activate local FSAP, leading to inhibition of PDGF-BB.     

In vitro preparation of amelogenin nanoparticles carrying nucleic acids

Amelogenin, a matrix protein involved in biomineralization of enamel, can self-assemble to form nanospheres in a pH-dependent manner. Nucleic acids (single-stranded, double-stranded, and plasmid DNA, as well as RNA) could be co-precipitated with amelogenin, demonstrating a strong binding of nucleic acids to amelogenin. The amounts of co-precipitated nucleic acids were analyzed and binding levels upto 90 μg DNA/mg amelogenin was achieved. The co-precipitation could also be carried out in a bacterial cell homogenate, and no bacterial proteins were found in the amelogenin aggregates, suggesting specificity for nucleic acid binding. Dynamic light scattering showed that amelogenin nanosphere structure is maintained upon DNA binding with an upto 2.6 nm increase in diameter. The reported binding of nucleic acids to amelogenin can be explored practically for nucleic acid separation.     

Substrate specificity and mode of action of the zinc-metallo nuclease from Physarum polycephalum

The alkaline zinc-metallo nuclease of Physarum polycephalum is an endonuclease with a high specificity for single-stranded nucleic acids. Single-stranded DNA was cleaved at least 6,000 times faster than double-stranded DNA under identical conditions. In the supercoil-induced single-stranded region of Form I PM2 DNA only a single nick was made. The nuclease showed nucleotide specificity. Poly(A), poly(I), and poly(dT) were preferentially hydrolyzed. Product analysis showed that it acted by an endonucleolytic mechanism: long polynucleotides were fragmented via intermediate length products to oligo- and mono-nucleotides with the phosphate group at the 5'-terminal position. Extensive similarities exist with the single-strand-specific nuclease S1 from Aspergillus. The zinc-metallo endonuclease from Physarum could be used as a similar probe for single-stranded nucleic acids at neutral or alkaline pH conditions.     

Fractionation with ethanol of nucleic acids from viroid-infected plants

A combination of high salt and low ethanol concentration allowed the fractionation of nucleic acids extracted from viroid-infected leaves. By adding 0.4-0.5 vol of ethanol to 1 vol of a solution in 2 M LiCl of nucleic acids (containing mainly DNA, 4S, 5S, 7S, and viroid RNAs), 85% of the DNA and 75% of the 4S RNA remained in solution, from where they could be recovered by increasing the ethanol concentration, whereas almost all 5S, 7S, and viroid RNAs precipitated. When this process was repeated three times a 95% elimination of the initial DNA and 4S RNA was achieved. The method can be of special interest in viroid purification considering that DNA and 4S RNA are the most abundant contaminants in the starting solution of nucleic acids. It is suggested that the highly ordered secondary structure of viroid RNA may be responsible for its particular behavior in the ethanol fractionation of nucleic acids.     

Labeling during cleavage of nucleic acids for their detection on DNA chips

A new and efficient strategy for labeling of nucleic acids prior to their hybridization on high density DNA chip has been developed. Our approach which combines the fragmentation and the labeling is based on the reactivity of the terminal phosphates of cleaved DNA and RNA fragments with a reporter molecule bearing aryldiazomethane group.     

Synthesis of nucleic acids in the Schwann cells as the early cellular response to nerve injury

Abstract— The early effect of mechanical injury of the sciatic nerve in cats and rats on the amount of nucleic acids and on the number of Schwann cells was studied. The content of RNA and DNA increased at both ends of a severed nerve as early as 2 hr after operation; at 8 hr and up to 24 hr, their level was about twice as high as at the beginning of experiment. The increase in nucleic acids seemed to be chiefly due to their biosynthesis in the Schwann cells although the possibility of a contribution of axonal RNA must also be taken into consideration. The division of Schwann cell nuclei was observed not earlier than 36 hr after operation. The injury stimulates the synthesis of nucleic acids not only in the Schwann cells directly damaged, but also in those from neighbouring internodes. The increase in DNA and RNA observed 24 hr after a fresh lesion had been made in a degenerating nerve was smaller than the increase observed 24 hr after cutting or crushing a normal nerve.     

The proto-Nucleic Acid Builder: a software tool for constructing nucleic acid analogs

The helical structures of DNA and RNA were originally revealed by experimental data. Likewise, the development of programs for modeling these natural polymers was guided by known structures. These nucleic acid polymers represent only two members of a potentially vast class of polymers with similar structural features, but that differ from DNA and RNA in the backbone or nucleobases. Xeno nucleic acids (XNAs) incorporate alternative backbones that affect the conformational, chemical, and thermodynamic properties of XNAs. Given the vast chemical space of possible XNAs, computational modeling of alternative nucleic acids can accelerate the search for plausible nucleic acid analogs and guide their rational design. Additionally, a tool for the modeling of nucleic acids could help reveal what nucleic acid polymers may have existed before RNA in the early evolution of life. To aid the development of novel XNA polymers and the search for possible pre-RNA candidates, this article presents the proto-Nucleic Acid Builder (https://github.com/GT-NucleicAcids/pnab), an open-source program for modeling nucleic acid analogs with alternative backbones and nucleobases. The torsion-driven conformation search procedure implemented here predicts structures with good accuracy compared to experimental structures, and correctly demonstrates the correlation between the helical structure and the backbone conformation in DNA and RNA.     

Nucleic acid oxidation in Alzheimer disease

Increasing evidence suggests that oxidative stress is intimately associated with Alzheimer disease pathophysiology. Nucleic acids (nuclear DNA, mitochondrial DNA, and RNA) are one of the several cellular macromolecules damaged by reactive oxygen species, particularly the hydroxyl radical. Because neurons are irreplaceable and survive as long as the organism does, they need elaborate defense mechanisms to ensure their longevity. In Alzheimer disease, however, an accumulation of nucleic acid oxidation is observed, indicating an increased level of oxidative stress and/or a decreased capacity to repair the nucleic acid damage. In this review, we present data supporting the notion that mitochondrial and metal abnormalities are key sources of oxidative stress in Alzheimer disease. Furthermore, we outline the mechanisms of nucleic acid oxidation and repair. Finally, evidence showing the occurrence of nucleic acid oxidation in Alzheimer disease will be discussed.     

绿豆子叶衰老期间核酸代谢的变化

萌发绿豆的子叶自然衰老期间,核酸含量降低,RNA降低的幅度比DNA大。电泳分析结果表明,子叶衰老期间细胞核主带DNA明显降低;而迁移慢的卫星带DNA变化不大。在RNA各组分中,18S rRNA从衰老前期就开始降低;25S rRNA和4～5S小分子RNA到衰老后期才缓慢下降。DNase和RNase活性在子叶整个衰老期间都明显升高,是导致核酸含量下降的主要原因。~3H-核苷掺入试验表明,核酸的合成速率在子叶衰老前期有所上升,到衰老后期又降低。poly(A)~ -mRNA含量在子叶开始衰老时明显上升。     

Interaction of resveratrol and genistein with nucleic acids

Resveratrol (RES) and genistein (GEN) are the dietary natural products known to possess chemopreventive property and also the ability to repair DNA damage induced by mutagens/carcinogens. It is believed that the therapeutic activity of these compounds could be primarily due to their interaction with nucleic acids but detailed reports are not available. We here explore the interaction of these drugs with nucleic acids considering DNA and RNA as a potential therapeutic target. The interaction of RES and GEN has been analysed in buffered solution with DNA [saline sodium citrate (SSC)] and RNA [tris ethylene diammine tetra acetic acid (TE)] using UV-absorption and Fourier transform infrared (FTIR) spectroscopy. The UV analysis revealed lesser binding affinity with nucleic acids at lower concentration of RES (P/D = 5.00 and 10.00), while at higher drug concentration (P/D = 0.75, 1.00 and 2.50) hyperchromic effect with shift in the lambda(max) is noted for DNA and RNA. A major RES-nucleic acids complexes was observed through base pairs and phosphate backbone groups with K = 35.782 M(-1) and K = 34.25 M(-1) for DNARES and RNA-RES complexes respectively. At various concentrations of GEN (P/D = 0.25, 0.50, 0.75, 1.00 and 2.50) hyperchromicity with shift in the lambda(max) from 260-->263 nm and 260--> 270 nm is observed for DNA-GEN and RNA-GEN complexes respectively. The binding constant (from UV analysis) for GEN-nucleic acids complexes could not be obtained due to GEN absorbance overlap with that of nucleic acids at 260 nm. Nevertheless a detailed analysis with regard to the interaction of these drugs (RES/GEN) with DNA and RNA could feasibly be understood by FTIR.     

Labeling During Cleavage of Nucleic Acids for Their Detection on DNA Chips

Abstract

A new and efficient strategy for labeling of nucleic acids prior to their hybridization on high density DNA chip has been developed. Our approach which combines the fragmentation and the labeling is based on the reactivity of the terminal phosphates of cleaved DNA and RNA fragments with a reporter molecule bearing aryldiazomethane group.     

Altered nucleic acid partitioning during phenol extraction or silica adsorption by guanidinium and potassium salts

Nucleic acids were found to partition into the phenol phase during phenol extraction in the presence of guanidinium at certain concentrations under acidic conditions. The guanidinium-concentration-dependent nucleic acid partitioning patterns were analogous to those of the nucleic acid adsorption/partitioning onto silica mediated by guanidinium, which implied that phenol and silica interact with nucleic acids through similar mechanisms. A competition effect was observed in which the nucleic acids that had partitioned into the phenol phase or onto the silica solid phase could be recovered to the aqueous phases by potassium in a molecular weight–salt concentration-dependent manner (the higher molecular weight nucleic acids needed higher concentrations of potassium to be recovered, and vice versa). Methods were developed based on these findings to isolate total RNA from Escherichia coli. By controlling the concentrations of guanidinium and potassium salts used before phenol extraction or silica adsorption, we can selectively recover total RNA but not the high molecular weight genomic DNA in the aqueous phases. Genomic DNA-free total RNA obtained by our methods is suitable for RT-PCR or other purposes. The methods can also be adapted to isolate small RNAs or RNA in certain molecular weight ranges by changing the salt concentrations used.     

Encapsulation of Nucleic Acids into Giant Unilamellar Vesicles by Freeze-Thaw: a Way Protocells May Form

Protocells are believed to consist of a lipid membrane and encapsulated nucleic acid. As the lipid membrane is impermeable to macromolecules like nucleic acids, the processes by which nucleic acids become encapsulated inside lipid membrane compartments are still unknown. In this paper, a freeze-thaw method was modified and applied to giant unilamellar vesicles (GUVs) and deoxyribonucleic acid (DNA) in mixed solution resulting in the efficient encapsulation of 6.4 kb plasmid DNA and similar length linear DNA into GUVs. The mechanism of encapsulation was followed by observing the effect of freeze-thaw temperatures on GUV morphological change, DNA encapsulation and ice crystal formation, and analyzing their correlation. Following ice crystal formation, the shape of spherical GUVs was altered and membrane integrity was damaged and this was found to be a necessary condition for encapsulation. Heating alone had no effects on DNA encapsulation, but was helpful for restoring the spherical shape and membrane integrity of GUVs damaged during freezing. These results suggested that freeze-thaw could promote the encapsulation of DNA into GUVs by a mechanism: the vesicle membrane was breached by ice crystal formation during freezing, DNA entered into damaged GUVs through these membrane gaps and was encapsulated after the membrane was resealed during the thawing process. The process described herein therefore describes a simple way for the encapsulation of nucleic acids and potentially other macromolecules into lipid vesicles, a process by which early protocells might have formed.     

Development of a sensitive and rapid nucleic acid assay with tetraphenyl porphyrinatoiron chloride by a resonance light scattering technique.

Based on the interaction between nucleic acids and tetraphenyl porphyrinatoiron chloride (FeTPPCl), a novel method for the determination of nucleic acids at the nanogram level has been developed. Under the optimum conditions, the weak resonance light scattering (RLS) intensity of FeTPPCl was greatly enhanced by nucleic acids and the enhanced RLS intensity was proportional to the concentration of nucleic acids in the range 0.02-2.8 microg/mL for calf thymus DNA, 0.05-3.3 microg/mL for fish sperm DNA and 0.07-4.5 microg/mL for yeast RNA. The detection limits (3sigma) were 2.9 ng/mL for calf thymus DNA, 3.9 ng/mL for fish sperm DNA and 5.2 ng/mL for yeast RNA. Almost no interference could be observed from proteins, nucleosides and most of the metal ions. The proposed method showed good reliability, sensitivity, rapidity and reproducibility when applied to the determination of nucleic acids in synthetic and biochemical samples. The time savings make this method suitable for large routine analyses.